The structure and extracellular regulation of the large latent TGFbeta complex

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

Transforming growth factor (TGF) beta is a powerful "growth factor" which is a messenger molecule that can direct processes in cells, for example telling them how much to grow, move or even whether they should live or die. These processes are crucial in maintaining normal tissue structure and function and are essential in human embryo developmental. The action of TGFbeta is controlled by large proteins found outside of the cell that bind to TGFbeta creating a tissue storage depot. This storage is needed for correct development, repair and maintenance of our tissues, such as the heart, lungs and skin.

The large proteins that bind to TGFbeta are called fibrillin and latent TGFbeta binding protein. Fibrillin forms fibres that are important for providing our tissues with elasticity such as large blood vessels like the aorta, lungs and skin. Symptoms of ageing associated with a loss of elasticity, for example skin wrinkles, hypertension and deterioration in vision, have been linked to degradation of fibrillin. Fibrillin and latent TGFbeta binding protein (LTBP) bind to TGFbeta, both these proteins are essential for the tissue storage of TGFbeta to occur correctly but there are currently no details of the fibrillin-LTBP-TGFbeta complex. Our limited knowledge regarding TGFbeta storage and the complexes they form presents a major obstacle to understanding TGFbeta function. The main aim of our work therefore is to understand the structure of the fibrillin-LTBP-TGFbeta complex which we believe will lead to an understanding of how TGFbeta storage occurs. We will determine the shape of LTBP when bound to TGFbeta, and how fibrillin and fibrillin fibres interact with this complex. Finally, we will discover how TGFbeta is released from this complex when it is needed and how these interactions underpin their important roles in tissue assembly and maintenance.

Understanding these molecular events for maintaining tissue elasticity could have significant health and economic benefits to the UK. Stiffening of the blood vessels and valves of the heart are major causes of heart disease which affects more than 6 million citizens in Europe each year. Heart disease has a huge economic impact, due to the high medical costs and work disability. In the eye, losing elasticity effects the ability to bend the lens (accommodation) which leads to the loss of up-close vision with age. This can be improved by wearing glasses but does not correct completely for this age-related deterioration in vision. Our research findings could be of future interest to the pharmaceutical industry in developing treatments to maintain the elasticity of these tissues. Effective treatment would significantly improve the quality of life of an ageing population.

Technical Summary

The transforming growth factor (TGF) beta family of growth factors play crucial roles in embryonic development, adult tissue homeostasis, the immune response and the pathogenesis of a wide-range of diseases from fibrosis to tumour invasion. The bioavailability of this class of growth factors is regulated by the extracellular matrix, providing a tissue store which is critical for tissue homeostasis and remodelling. Two molecules are essential for the sequestration of TGFbeta in the matrix: fibrillin and latent TGFbeta binding protein (LTBP). Fibrillin microfibrils are extensible polymers that are critically important in maintaining the integrity of tissues such as blood vessels, lung and skin. Fibrillin binds to LTBP-1 which in turn is covalently attached to TGFbeta. The expression of LTBP-1 is co-regulated with TGFbeta and together they are secreted as the large latent TGFbeta complex (LLC). The lack of knowledge regarding TGFbeta regulators and the complexes they form presents a major hurdle to understanding TGFbeta bioavailability.

The aim of our work therefore is to define the structure and organisation of the large latent TGFbeta complex and its interaction with fibrillin, leading to an understanding of how TGFbeta sequestration via fibrillin is controlled in tissue assembly and homeostasis. Specifically, we will determine the nanoscale structure of the LLC and how the LLC interacts with fibrillin and fibrillin microfibrils. We will determine the molecular events surrounding release of TGFbeta from the LLC, mediated by integrins and proteases, and the role fibrillin plays in this process. We will use a multi-technique approach of structural, biochemical and biophysical techniques including electron microscopy, solution X-ray scattering, multiangle light scattering, analytical ultracentrifugation, scanning TEM mass mapping, atomic force microscopy and binding analyses (surface plasmon resonance and dual polarisation interferometry).

Planned Impact

We anticipate that the results gained from this study will be of both significant intellectual and clinical benefit as the project will deliver high-quality biochemical research on a fundamental process in mammalian biology. In particular, this work is relevant to the BBSRC Strategic Research Priority "Ageing research: lifelong health and wellbeing" because of the vital roles fibrillin and the large latent TGFbeta complex play in maintaining the normal structure and function of tissues such as the skin, heart, lungs and eyes.

This work will provide novel insights into a molecular mechanism relevant to tissue assembly, in particular elastic tissues. This proposal is to undertake basic science underpinning the regulation of homeostatic events in tissues, but our research findings could be of future interest to the pharmaceutical industry in developing treatments to maintain the elasticity of tissues. We will utilise the Faculty Research Support Managers, part of whose remit is to facilitate interactions with industry and University of Manchester Intellectual Property (UMIP) to identify outcomes with commercial potential.

The results of this study will be of academic benefit to a range of research communities including connective tissues, development, growth factor and structural biology research communities as outlined in the academic beneficiaries section. We will disseminate the results of this research through participation at relevant conferences and through publications in peer-review journals as outlined in the previous section. We are also committed to public engagement in science. For example, the Faculty of Life Sciences (FLS) is active in promoting the communication of science to the public (in which the applicants' groups participate). Initiatives include reporting research breakthroughs in the local, national and international press via the Faculties Media Relations Office and schools outreach work (e.g. curriculum enrichment in the form of career advice, practical classes, and workshops). In this regard, the electron microscopy facility in FLS hosts regular visits from schools for pupils in years 11-13 and the EM facility runs tours during the annual Faculty of Life Sciences Community Open Day in which all lab members participate.

Training and development of Helen Troilo, the named RA, in new techniques such as cryo-EM will enhance her research career. FLS has embraced training and career development for all categories of staff and current support available to PDRAs for professional development includes monthly training bulletins, one-to-one advice and guidance and bespoke workshops. Recent workshops have included: "Planning a Fellowship", "Grant Reviewing", "Academic CV Writing" and a "Careers Day". These workshops have aimed to develop a range of skills including career planning, networking, project management, team working, critical peer review, communication and self awareness.

Publications

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Lockhart-Cairns MP (2019) Internal cleavage and synergy with twisted gastrulation enhance BMP inhibition by BMPER. in Matrix biology : journal of the International Society for Matrix Biology

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Schmelzer CEH (2019) Lysyl oxidase-like 2 (LOXL2)-mediated cross-linking of tropoelastin. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

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Troilo H (2016) Structural characterization of twisted gastrulation provides insights into opposing functions on the BMP signalling pathway. in Matrix biology : journal of the International Society for Matrix Biology

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Yeo GC (2017) Targeted Modulation of Tropoelastin Structure and Assembly. in ACS biomaterials science & engineering

 
Description Our studies on LTBP1 show it is an elongated molecule with flexible regions. LTBP1 can self-assemble via N-ter and C-ter interactions to form short filamentous structures. This self assembly is enhanced by heparan sulphate binding. We have also showed that LTBP1 can be cross-linked to fibrillin by tissue transglutaminase to stabilise higher order structures. Cross-linking can occur in the presence of bound TGFbeta but not when LTBP1 has self-assembled indicating that these assemblies have different roles. Furthermore, soluble fibrillin fragments do not provide latency to the latent TGFbeta complex but enhance its activation by directing the complex to the cell surface.
Exploitation Route Our research findings could be of future interest to the pharmaceutical industry in developing ways to stabilise or inhibit activation of latent TGFbeta in treatment of fibrillinopathies such as Marfan syndrome. In particular the knowledge that fibrillin stabilises the large latent TGFbeta complex via cross-linking by transglutaminase could be beneficial in considering therapeutic targets.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description Dual functionalities of the LTBP family in matrix assembly and growth factor regulation
Amount £453,465 (GBP)
Funding ID BB/S015779/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2019 
End 06/2022
 
Description American Society for Matrix Biology Annual Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Type Of Presentation poster presentation
Geographic Reach International
Primary Audience Other audiences
Results and Impact A talk was presented at the ASMB meeting in Las Vegas, USA in October 2018 by Clair Baldock.

A talk was presented at the ASMB meeting in St Petersburg, Florida in November 2016 by Clair Baldock and a poster presented by Dr Michael Lockhart.

A poster was presented at the ASMB meeting in Cleveland, USA in October 2014 by PDRA Dr Christopher Bayley. His poster attracted many visitors who discussed his findings and had suggestions for collaboration.

Sparked a new collaboration to explore ideas further.
Year(s) Of Engagement Activity 2014,2016,2018
URL http://www.asmb.net/Pastmeetings.php
 
Description Chair of Elastin, Elastic Fibers and Microfibrils Gordon Research Conference 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Chair of the Elastin, Elastic Fibers and Microfibrils Gordon Research Conference, July 2017 at the University of New England, Maine USA. 116 attendees including academic, industry and government employees. In addition to organising and chairing the meeting, fundraising of >$80,000 was achieved including an NIH grant. Four members of my lab presented posters, one won the poster prize, one member of my lab gave a short-talk and another chaired a session.
Year(s) Of Engagement Activity 2017
URL https://www.grc.org/elastin-elastic-fibers-and-microfibrils-conference/2017/
 
Description Daresbury Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact My laboratory hosted a stand at Daresbury Laboratories STFC for a week in July 2016 to illustrate research performed through STFC (ie at synchrotrons). We had ~500 pupils per day visit the stand from Key Stage 2 to Key Stage 5 and at the weekend the site was open to the public attracting thousands of visitors.
Year(s) Of Engagement Activity 2016
URL http://www.stfc.ac.uk/public-engagement/activities-for-the-public/visit-daresbury-laboratory/daresbu...
 
Description Elastin and Microfibrils Gordon Research Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other audiences
Results and Impact Biannual invited talk at the Elastin GRC for PI Clair Baldock since 2003. Talk facilitated discussion of the use of SAXS for other elastic fibre proteins.

Elected vice-chair of GRC for 2015 (chair in 2017).
Collaboration on SAXS of tropoelastin has led to 2 PNAS, 1 JBC and 1 Matrix Biology papers to date.
Talks led to invitation to visit two Institutions in Canada (Sick Kids Hospital, Toronto and Simon Fraser University, Vancouver) and to Shriners Hospital in Portland in 2011.
Year(s) Of Engagement Activity Pre-2006,2006,2007,2009,2011,2013,2015,2017,2019
 
Description European Elastin Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact PI Clair Baldock and group members attend biannual European Elastin Meetings to network with European community and raise profile of research.

Invitation to participate in a Marie Curie Sklodowska ITN network and elected vice-chair of the Elastin Gordon conference for 2015 (Chair in 2017).
Year(s) Of Engagement Activity 2008,2010,2012,2014,2016,2018
 
Description German Connective Tissue Society Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited Speaker at the German Connective Tissue Society Meeting in Cologne March 2017 and January 2019.
Year(s) Of Engagement Activity 2017,2019
URL http://matrixbiologie.de/JahrestagungKoeln/CologneDGMB-Index.html
 
Description Matrix Biology Europe 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talks and posters stimulated discussion.

Mukti Singh (BBSRC DTP PhD student) was awarded a poster prize.

After talks I was asked to talk at National Societies including the German Connective Tissues Society Meeting in Cologne in 2011 and the French Connective Tissue Society Meeting in 2012.
Led to participation in an application for a Marie Curie Sklodowska ITN network.
Year(s) Of Engagement Activity 2010,2012,2018
 
Description S4SAS Workshop and Meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact In 2010, PI Clair Baldock gave an invited seminar at the inaugral S4SAS meeting.
In 2012, PDRA Dr Chris Bayley attended a training workshop in small angle X-ray scattering to gain skills in this technique. He also presented a poster to the user community for discussion and advice.
In 2014, PhD student then PDRA Dr Helen Troilo gave a talk and poster and was awarded the poster prize for the meeting. Helen subsequently was award the runner up proze for the Diamond PhD Investigator of the year Award 2014.

CB invited on the steering committees for Diamond's new protein scattering beamline B21 and software.
New methods were suggested in discussion and use of the OPPF facility which we followed up.
Year(s) Of Engagement Activity 2010,2012,2014